Topsin-M: the new benomyl for mycorrhizal-suppression experiments

The fungicide benomyl was the most commonly used biocide for both field and greenhouse experiments in which arbuscular mycorrhizal fungal (AMF) suppression is desired. Unfortunately benomyl is no longer manufactured and therefore is not available for experimental use and no fungicide has been proposed as a successful alternative for experimentally suppressing mycorrhizal fungi. In this study we examined the potential for the fungicide Topsin M (topsin) to suppress mycorrhizal symbiosis in both field and greenhouse experiments. Topsin reduced AMF colonization of the obligately mycotrophic, warm-season grass Andropogon gerardii with a large and significant reduction in plant biomass production. Topsin reduced AMF colonization of the facultatively mycotrophic, cool-season grass Pascopyron smithii but did not significantly reduce biomass production. Fertilization with nitrogen and phosphorus was able to compensate for reductions in biomass due to the application of fungicide because biomass production of plants that received topsin fungicide was not significantly different from fertilized controls not receiving topsin. While we are not advocating that topsin fungicide is a universal mechanism for mycorrhizal-suppressed controls, in systems where benomyl was found to be successful topsin appears to be a useful, available and successful alternative.     

Benomyl: A broad spectrum fungicide for use in plant cell and protoplast culture

The systemic fungicide methyl-1-(butylcarbamoyl)-2-benzimidazole carbamate (benomyl), is a broad spectrum fungicide. Benomyl at concentrations up to 50 mg/l does not inhibit the growth of suspension cultures ofNicotiana tabacum, Datura innoxia, Daucus carota, Glycine canescens, andSolanum tuberosum nor growth ofN. tabacum orN. plumbaginifolia protoplasts if benomyl is dissolved by autoclaving or boiling. Addition of benomyl dissolved in dimethyl sulfoxide results in a visible toxicity. Benomyl, at 6.25–50 mg/l preventsPenicillium spp. growth in both protoplast and cell cultures and can be used to remove fungal contaminates after one to three transfers without visibly retarding plant cell growth. Due to the broad spectrum of fungicidal activity, and nontoxicity at high concentrations when dissolved by boiling or autoclaving, benomyl can be used effectively to control or prevent fungal contamination in plant cell and protoplast cultures.     

Benomyl and mancozeb as fungicides in soil studies of Azotobacter and Beijerinckia

Summary Routine media containing 0.005% each of benomyl and mancozeb for the enumeration and isolation of Azotobacter and Beijerinckia in soil studies are described. The incorporation of these fungicides in routine media was found to be more effective in inhibiting fungal contaminants than the use of cycloheximide.     

The effect of benomyl on growth and ultrastructure of two isolates of Phytophthora infestans from Egypt

The effect of benomyl as a fungicide on the growth rate and ultrastructure of two isolates (P1319 and P623) of Phytophthora infestans is compared. Benomyl caused a concentration-dependent inhibition of the mycelial growth of both isolates. The isolate P1319 was found to be more sensitive to benomyl than the isolate P623. Ultarstructural studies confirmed these observations. The hyphae of isolate P1319 subjected to 100 and 500 ppm benomyl showed more severe changes in the cytoplasm than those of isolate P623. An increase in lipid bodies and vacuoles in the hyphal cytoplasm was the characteristic phenomenon after treatment with benomyl, particularly at a concentration of 500 ppm.     

Effect of benomyl on the growth and lipid composition ofTrichoderma koningii

The effect of the fungicide benomyl on growth and lipid composition ofTrichoderma koningii was investigated. The fungal growth was strongly inhibited in the presence of 1 and 2 mg/L benomyl while lower concentrations (0.1 and 0.5 mg/L) increased the fungal biomass through the stimulation of mycelial branching. The total lipid and the total neutral lipid were decreased, while the total phospholipid was enhanced in benomyl-treated mycelia. Important quantitative changes were detected in the proportions of fatty acids, neutral lipid fractions (decrease of free sterols, diacylglycerols and free fatty acids and increase of triacylglycerols and sterol esters) and phospholipid constiuents (decrease of phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine and increase of phosphatidylglycerol and phosphatidylinositol). The unsaturation index of the identified fatty acids was increased with increasing benomyl concentration.     

Evaluation of the effects of chemical versus biological control on Botrytis cinerea agent of gray mould disease of strawberry

This study investigates on effects of four fungicide and six isolate from Trichoderma and Gliocladium on Botrytis cinerea agent grey mold of strawberry under library and greenhouse condition. The effect of four fungicides i.e. benomyl, dichlofluanid, captan and triadimenol on B. cinerea was studied in the laboratory condition by method mixed poison to culture medium. It was shown that the fungicide including benomyl, triadimenol, dichlofluanid and captan were able to inhibit mycelial growth of B. cinerea on PDA plate with EC50 of 0.16, 1.42, 3.40 and 7.73 ppm respectively. These fungicides delayed myceliogenic germination of sclerotia at 1000 ppm, while exhibiting no fungicidal effect. Moreover, the antagonistic effects of six fungi including Trichoderma koningii (T21), T. viride (T4), T. harzionum (T5), T. viride (T2), G. virens (G2), G. virens (G8) on B. cinerea were assessed. This assessment was done under library condition and its results as follows: The antagonistic mechanism occurred through branching at the end of B. cinerea hyphae, hyphal contact, coiling, vacuolization and lyses. Volatile metabolites of T. koningii (T21) and non-volatile metabolites of G. virens (G2 and G8) and T. koningii (T21) caused maximum inhibition of the fungal growth. Trichoderma spp and G. virens were able to colonize and sporulate on sclerotia and caused their lysis within 7-21 days. In greenhouse, a completely randomized design with 11 treatments (4 chemical and 6 biological and one untreated control) each replicated five times were used for the comparison. Greenhouse studies revealed that application of fungicides i.e. captan, dichlofluanid, triadimenol and benomyl reduces disease severity by 42, 45, 48 and 52% respectively. The fungal antagonists reduce the grey mold disease severity between 5-42%. All treatments caused a decline in post harvest disease, as the most effective treatment of chemical control was benomyl with 68.33% and for the biological treatment this was T. koningii (T21) with 56%.     

Pollen germination, tube growth and morphology, and microtubule organization after exposure to benomyl

The inhibitory effects of benomyl on pollen tube growth have received little attention, particularly at the microscopic and immunohistochemical levels. Pollen germination and tube growth in the presence of benomyl were evaluated in Tradescantia virginiana to investigate the effects of this fungicide on pollen germination rate, tube growth and morphology, and microtubule (Mt) organization. Benomyl was incorporated in germination media at 0,480, 600 or 720 mg 1⁻¹. Inhibition of pollen germination, cytoplasmic streaming, and tube elongation were associated with benomyl treatments. Benomyl also induced abnormal pollen tube morphology and Mt organization. Compared to controls, Mts in the treated tubes were characteristically fewer in number, fragmented, sinuous and increasingly disorganized. At the two highest benomyl concentrations, Mts were considerably fewer or absent in apical/subapical regions of the pollen tubes. This work verifies that benomyl incorporated into germination media at concentrations lower than recommended field rates inhibit pollen germination and tube growth, and that the effects are associated with alterations of Mt organization.     

PHYTOTOXICITY OF FUNGICIDES AND BACTERICIDES IN ORCHID CULTURE MEDIA

A number of fungicides, bactericides and dual-action substances were screened for phytotoxicity in vitro using Cattleya aurantiaca and Stanhopea occulata (Orchidaceae) seedlings. Of the 25 compounds screened, ten (each at only one concentration) were employed in nine different combinations for nonsterile in vitro cultures of Cattleya aurantiaca seedlings: Amphotericin B, 10 ppm; Benlate, 50 ppm; Dowicide, 5 ppm; Ethirimol, 50 ppm; Gentamicin, 50 ppm; Nystatin, 25 ppm; PCNB, 100 ppm; Penicillin G, 100 ppm; Sodium omadine, 5 ppm; and Vancomycin, 50 ppm. All nine combinations prevented contamination, but had different effects on seedling development.     

Movement and containment of microbial contamination in the nutrient mist bioreactor

Summary In vitro plant cultures tend to get contaminated easily with bacteria and fungi because they are grown for long times in sugar-rich media. Contamination of bioreactors is particularly problematic as larger volumes entail larger losses. To study the movement and develop subsequent control of contaminants in the mist bioreactor, the spore-forming microbes Penicillium chrysogenum and Bacillus subtilis were deliberately inoculated into three possible locations in the reactor: the growth chamber (GC), the medium reservoir (R), or the mist-generating chamber (MG). Compared to inoculation into either R or MG regions, the growth of P. chrysogenum inoculated into the GC required 3 more days (c. 60% more time) to move throughout the rest of the reactor. In contrast, regardless of where B. subtilis was inoculated (GC, R, or MG), it took 7d to contaminate the entire system. The movement of filamentous fungi and bacteria seems to follow the same route of contamination throughout this reactor. Once visibly present in the reactor, neither contaminant was controllable by addition of the biocide, Plant Preservative Mixture (PPM). Both microbes were completely inhibited if PPM was added to the MG at the time of inoculation and then again 2-d post-inoculation of plants. Reactors were fun for 3 wk. Plants remained free of contamination. These results will prove useful in the implementation of large-scale in vitro culture systems.     

Strains of Colletotrichum coffeanum, the causal agent of coffee berry disease, tolerant to benzimidazole compounds in Kenya

The detection of Colletotrichum coffeanum tolerant to methyl ester of benzimidazole 2-carbamic acid (carbendazim) and a related benzimidazole compound, cypendazole, followed increases in levels of coffee berry disease observed on Coffea arabica in experimental plots sprayed for 2 yr with these compounds. Sporulation by the pathogen on naturally infected berries removed from carbendazim-, cypendazole- or benomyl-sprayed plots was not checked by a further application of 0–05 % (a.i.) of any of the compounds. Nearly all the isolates from these berries were capable of some growth on agar media containing 1000 ppm (a.i.) of either carbendazim or cypendazole. However, only a few could tolerate 1000 ppm of benomyl and the inability of this compound to reduce sporulation on berries infected with tolerant strains was presumably due to its rapid conversion to carbendazim within the host tissue. Less than 1 ppm of carbendazim, cypendazole or benomyl was needed to give 50% inhibition of conidia of the normal strain. Against the most tolerant strains, however, the LD 50 was > 100 ppm of carbendazim and about 30 ppm of benomyl. Whether isolated from unsprayed or benzimidazole-sprayed plots, all isolates of Colletotrichum acutatum, a saprophytic cohabitant of lesions initiated on berries by C. coffeanum, showed the highest degree of tolerance to benzimidazole compounds. No tolerance of either fungus to the ‘conventional’ fungicide captafol was detected.     

The effects of fungicide benomyl (benlate) on growth and mitosis in onion (Allium cepa L.) root apical meristem

In this study, the effects of benomyl, a systemic fungicide were investigated in the mitotic cell division in onion (Allium cepa) root tip cells during germination. For this aim, different concentrations (1, 2, 5, 10 and 20 mM) of benomyl solutions were used. All the concentrations used caused several abnormalities in mitotic cell divisions and the mitotic frequency in the onion root tip cells decreased as the concentration of benomyl solution increased. Based on our findings, it is reported that benomyl has some negative effects on mitotic divisions in onion root tip cells.     

Effects of benomyl on reproduction and population structure of enchytraeid oligochaetes (Annelida)—Sublethal tests on agar and soil

• 1.1. The toxicity of the fungicide benomyl to terrestrial enchytraeid species was tested under different conditions.
• 2.2. Despite a relatively low acute response even to higher concentrations in agar media, sensitive effects were observed for cocoon production and hatching success at the recommended concentration for agricultural application (5.9 ppm in the test medium).
• 3.3. These sublethal effects could be reconciled with population tests in larger quantities of soil: lower abundance was due to a very low number of juveniles in the benomyl-treated cultures.

     

Benomyl: Effectiveness against the microsporidian Nosema heliothidis in the corn earworm,Heliothis zea

The fungicide benomyl was studied as a possible antimicrobial agent for obtaining Nosema heliothidis-free laboratory colonies of Heliothis zea. Newly hatched, transovarially infected larvae were placed on artificial diets containing 250, 500, or 1000 ppm benomyl. While late-stage larvae were found to be free of Nosema spores, low-level infections were found in pupae and newly emerged adults. The reduced intensity of infection in adults reared as larvae on treated diets was not correlated with a significant reduction in the efficiency of transovarian transmission. The chemical effect of benomyl was manifested by aberrant spores and vegetative stages and a rapid reduction in the number of microsporidian stages. However, small, isolated centers of infection in various host tissues resulted in a rapid resurgence of the microsporidiosis in pupae and adults. Thus, at the concentrations tested, benomyl was not effective in eliminating infection by N. heliothidis in H. zea. A discussion of the necessity for careful evaluation of the apparent suppression of microsporidioses by antimicrobial agents is also presented.     

Biotoxic effects of Sumisclex and Carbomar fungicides on four Aspergillus species isolated from the rhizosphere of some Taif plants

Abstract

The antifungal activities of two systemic fungicides (Sumisclex and Carbomar) on four rhizospheric Aspergillus species (A. candidus, A. sydawi, A. niger1 and A. niger2) were studied. Also the phytotoxicity against some saudian seeds was analysed. Results indicated that Sumisclex fungicide was more effective on the growth of A. candidus and A. sydawi under laboratory conditions (the IC₅₀ of 3 and 4 ppm were recorded, respectively) than Carbomar where an IC₅₀ of 11 ppm for A. candidus and 35 ppm for A. sydawi were observed. The growth of the two strains of A. niger was completely inhibited by Carbomar even at the lowest 10 ppm concentration. On the other hand results revealed that phytotoxicity was apparently linked with the type of the fungicide and its concentration. However, the root system was more sensitive to the tested fungicides especially at higher concentrations.     

The effectiveness of fungicides used against Claviceps purpurea attacking male-sterile barley in field trials

Sprays of benomyl applied during the flowering period to artificially inoculated barley heads were shown to be effective against ergot disease of male-sterile barley. Under natural conditions, using germinating ergots as a source of ascospore inoculum, the effectiveness of benomyl was reduced — the reasons for this are discussed. Timing of fungicide application was critical as none of the fungicides tested showed marked eradicant properties. Sprays of benomyl just before and at maximum flower opening were best.     

Trace elements in the otoliths and dorsal spines of albacore tuna (Thunnus alalunga, Bonnaterre, 1788): An assessment of the effectiveness of cleaning procedures at removing postmortem contamination

Trace element analysis or “elemental fingerprinting” is widely used in stock structure analyses. Postmortem contamination of bony structures can confound the results of microconstituent studies or introduce an additional source of noise to the data, thus reducing the ability of the technique to detect real variation in trace element concentrations. Despite the potential for postmortem contamination during sample preparation, the effectiveness of the procedures used to remove potential contaminants from sectioned otoliths and other calcareous structures prior to laser ablation inductively coupled plasma mass spectrometry (LA ICP-MS) has not previously been addressed. Otoliths and dorsal spine sections of albacore tuna (Thunnus alalunga) collected from the North East Atlantic Ocean and the Mediterranean Sea were deliberately contaminated prior to analysis of trace element composition using LA ICP-MS. The effectiveness of three cleaning treatments (rinsing in ultrapure water, 30% hydrogen peroxide and ultrapure 5% nitric acid) at removing this postmortem contamination were compared. Magnesium and strontium were relatively robust to postmortem effects when exposed to contamination at concentrations of 50 ppm and 200 ppm respectively. Soaking in a solution containing Mn, Cs and Ba (50 ppm) caused a marked increase in the detected concentration of each element in both structures. Translucent bands in both structures were more susceptible to contamination. Rinsing in ultrapure water or hydrogen peroxide was not effective at removing Mn, Cs and Ba contamination from either calcareous structure. Washing the otoliths and spines in nitric acid successfully removed postmortem contaminants.The removal of otoliths from tuna damages the appearance of the fish and has an adverse effect on market value. However spines are easily removed, do not affect the appearance or value of the fish and are the most commonly used structure for age determination. A weak but significant correlation was observed between Ba in opaque zones in otoliths and dorsal spines. All other spine to otolith correlations were not significant. The results do not provide support for the use of spines as an alternative to otoliths in trace elemental analyses.     

Development of an In vitro pod culture technique for young pods of Phaseolus vulgaris L.

Summary A research program to develop an in vitro culture technique adapted to very immature Phaseolus zygotic embryos indicated that osmolality within young pods in vivo ranged from 580 to 350 mosm during the first 10 d after pollination. Different culture techniques for 2-d-old Phaseolus pods are described using a modified Phillips medium for maturation. The application of high and variable osmolality conditions, similar to what is observed in vivo, during pod culture (1 wk) and before extracting the embryos, gave the best results in terms of ovule and embryo development. To solve contamination problems affecting pod development during these investigations, a sterilizing technique was tested combining various concentrations of polyoxyethylenesorbitan monolaurate (Tween 20) and a broad-spectrum industrial biocide, Plant Preservative Mixture (PPM). The PPM sterilizing method totally eliminated pod contamination and did not affect ovule growth, but significantly reduced the embryo germination rate.     

Legionella in industrial cooling towers: monitoring and control strategies

Aims: Legionella contamination of industrial cooling towers has been identified as the cause of sporadic cases and outbreaks of legionellosis among people living nearby. To evaluate and control Legionella contamination in industrial cooling tower water, microbiological monitoring was carried out to determine the effectiveness of the following different disinfection treatments: (i) continuous chlorine concentration of 0·01 ppm and monthly chlorine shock dosing (5 ppm) on a single cooling tower; (ii) continuous chlorine concentration of 0·4 ppm and monthly shock of biocide P3 FERROCID 8580 (BKG Water Solution) on seven towers. Methods and Results: Legionella spp. and total bacterial count (TBC) were determined 3 days before and after each shock dose. Both strategies demonstrated that when chlorine was maintained at low levels, the Legionella count grew to levels above 10⁴ CFU l^?1 while TBC still remained above 10⁸ CFU l^?1. Chlorine shock dosing was able to eliminate bacterial contamination, but only for 10–15 days. Biocide shock dosing was also insufficient to control the problem when the disinfectant concentration was administered at only one point in the plant and at the concentration of 30 ppm. On the other hand, when at a biocide concentration of 30 or 50 ppm was distributed throughout a number of points, depending on the plant hydrodynamics, Legionella counts decreased significantly and often remained below the warning limit. Moreover, the contamination of water entering the plant and the presence of sediment were also important factors for Legionella growth. Conclusions: For effective decontamination of outdoor industrial cooling towers, disinfectants should be distributed in a targeted way, taking into account the possible sources of contamination. Significance and Impact of the Study: The data of the research permitted to modify the procedure of disinfection for better reduce the water and aerosol contamination and consequently the exposure risk.     

The effect of the systemic fungicide benomyl on survival and reproduction of the bird cherry aphid (Rhopalosiphum padi)

Weight gain and intrinsic rate of population increase (r_m) of Rhopalosiphum padi were measured on wheat plants given soil drenches of benomyl at various concentrations. Reduced weight gain occurred with concentrations as low as 3.125 ppm. Mortality of aphids significantly exceeded control mortality at 25 ppm, and no aphids survived concentrations above 50 ppm. Development time was extended at 12.5 ppm and above. Reductions in fecundity and r_m became statistically significant by 25 ppm. Topical application of benomyl at 100 ppm or above increased aphid mortality, which reached 97% at 400 ppm. Weight gain of aphids was also reduced by topical application at 100 ppm. A factorial experiment showed absence of interaction between the effects on aphid weight of soil drench application to plants and topical application to aphids.     

Combined application of Pseudomonas fluorescens strain LRB3W1 with a low dosage of benomyl for control of cabbage yellows caused by Fusarium oxysporum f. sp. conglutinans

An antagonistic bacterium, Pseudomonas fluorescens strain LRB3W1, inhibited the fungal growth of Fusarium oxysporum f. sp. conglutinans in vitro and suppressed cabbage yellows caused by F. oxysporum f. sp. conglutinans. Under glasshouse conditions, the bacterium survived at ca. 10⁶-10⁷ CFU g^-1 in the cabbage rhizosphere for 4 weeks after the initial application. The chemical fungicide, benomyl, did not suppress the disease severity at low concentration (1 or 10 µg mL^-1). However, the disease severity was decreased by the combined application of a low dosage of benomyl with strain LRB3W1. Combined application of a low dosage of benomyl with strain LRB3W1 was more effective than treatment with the bacterium alone. The survival of strain LRB3W1 was not influenced by the presence of benomyl. This combined use of the biocontrol agent, strain LRB3W1, and a fungicide, benomyl, should be an attractive approach for suppressing cabbage yellows in sustainable agriculture because of the reduced chemical dosages needed for disease management.